Xylitol dehydrogenase of acetic acid bacteria and gene thereof

ABSTRACT

Xylitol is produced by allowing xylitol dehydrogenase or cells instoduced with a DNA coding for xylitol dehydrogenase, which is a protein of the following (A) or (B) to act on D-xylulose, and collecting produced xylitol:  
     (A) a protein which has the amino acid sequence of SEQ ID NO: 4;  
     (B) a protein which has the amino acid sequence of SEQ ID NO: 4 including substitution, deletion, insertion, addition, or inversion of one or several amino acids, and has xylitol dehydrogenase activity.

TECHNICAL FIELD

[0001] The present invention relates to a novel xylitol dehydrogenase ofacetic acid bacteria, gene coding for the same, method for producingxylitol dehydrogenase, and method for producing xylitol. Xylitol isuseful in the fields of food industry, drug industry and the like.

BACKGROUND ART

[0002] Xylitol, which is a naturally occurring sugar alcohol, is apromising low-calorie sweetener because it has lower calories butexhibits comparable sweetness compared with sucrose. In addition,because of its anti-dental caries property, it can be a dental cariespreventive sweetener. Furthermore, because xylitol does not elevateglucose level, it has been utilized for fluid therapy in the treatmentof diabetes mellitus. For these reasons, it is expected that the demandof xylitol will increase in future.

[0003] The current industrial production of xylitol mainly relies onhydrogenation of D-xylose as disclosed in U.S. Pat. No. 4,008,285.D-Xylose used as a raw material is obtained by hydrolysis of plantmaterials such as trees, straws, corn cobs, oat hulls and otherxylan-rich materials.

[0004] However, such D-xylose produced from hydrolysis of plantmaterials suffers from a drawback that it is rather expensive, and it isarisen from high production cost. For example, the low yield of thehydrolysis treatment of plant materials leads to low purity of theproduced D-xylitol. Therefore, the acid used for the hydrolysis and thedyes must be removed by ion exchange treatment after the hydrolysistreatment, and the resulting D-xylose must be further crystallized toremove other hemicellulose saccharides. In order to obtain D-xylosesuitable for foodstuffs, further purification would be required. Suchion exchange treatment and crystallization treatment invite the increaseof production cost.

[0005] Therefore, several methods for producing xylitol have beendeveloped, which utilize readily available raw materials and generatelittle waste. For example, there have been developed methods forproducing xylitol utilizing other pentitols as a starting material. Oneof such readily available pentitols is D-arabitol, and D-arabitol can beproduced by using yeast (Can. J. Microbiol., 31, 1985, 467-471; and J.Gen. Microbiol., 139, 1993, 1047-54).

[0006] Thus, several methods for producing xylitol that utilizeD-arabitol as a raw material have been developed. One method has beenreported in Applied Microbiology, 18, 1969, 1031-1035, whereinD-arabitol is produced from glucose by fermentation using Debaryomyceshansenii ATCC20121, then converted into D-xylulose using Acetobactersuboxydans, and the D-xylulose is converted into xylitol by the actionof Candida guilliermondii var. soya.

[0007] EP 403 392A and EP421 882A disclose methods which compriseproducing D-arabitol by fermentation using an osmosis-resistant yeast,then converting D-arabitol into D-xylulose using a bacterium belongingto the genus Acetobacter, Gluconobacter, or Klebsiella, forming amixture of xylose and D-xylulose from the D-xylulose by the action ofglucose (xylose) isomerase, and converting the produced mixture ofxylose and D-xylulose into xylitol by hydrogenation. There is alsodisclosed the production of xylitol comprising preliminarilyconcentrating xylose in the mixture of xylose and D-xylulose andconverting the concentrated xylose into xylitol by hydrogenation.

[0008] While those methods for the production of xylitol utilizingD-arabitol as a starting material mentioned above can produce xylitolwith a relatively high yield, however, they suffer from a drawback thatthey requires multiple reaction steps, and hence the processes shouldbecome complicated. Therefore, they have not been economicallyacceptable.

[0009] On the other hand, breeding of xylitol fermenting microorganismshas been attempted by using genetic manipulation techniques.International Publication WO94/10325 discloses production of xylitolfrom glucose through fermentation by using a recombinant microorganismobtained by introducing an arabitol dehydrogenase gene derived from abacterium belonging to the genus Klebsiella and a xylitol dehydrogenasegene derived from a bacterium belonging to the genus Pichia into anarabitol fermenting microorganism (yeast belonging to the genus Candida,Torulopsis, or Zygosaccharomyces).

[0010] However, such breeding of xylitol fermenting microorganisms byusing genetic manipulation techniques as mentioned above is notconsidered to be completed as a practical means.

[0011] By the way, xylitol dehydrogenase is an enzyme that catalyzes thereaction producing xylitol from xylulose, and its presence has beenknown in various organisms. For example, there has been known thepresence of xylitol dehydrogenase in yeast species such as Pichiastipitis (J. Ferment. Bioeng., 67, 25 (1989)), Pachysolen tannophilus(J. Ferment. Technol., 64, 219 (1986)), Candida shehatae (Appl. Biochem.Biotech., 26, 197 (1990)), Candida parapsilosis (Biotechnol. Bioeng.,58, 440 (1998)), Debaryomyces hansenii (Appl. Biochem. Biotech., 56, 79(1996)), and Pullularia pullulans (An. Acad. Brasil. Cienc., 53, 183(1981)), filamentous bacteria such as Aspergillus niger (Microbiology,140, 1679 (1994)) and Neurospora crassa (FEMS Microbiol. Lett., 146, 79(1997)), algae such as Galdieria sulphuraria (Planta, 202, 487 (1997)),bacteria such as Morgannela morganii (J. Bacteriol., 162, 845 (1985)),and the like.

[0012] As for the xylitol dehydrogenase gene, there have been reportednucleotide sequences of the gene derived from Pichia stipitis (FEBSLett., 324, 9 (1993)) and Morgannela morganii (DDBJ/GenBank/EMBLaccession No. L34345).

[0013] However, xylitol dehydrogenase derived from acetic acid bacteriaand its gene have not been known so far even for their presence itself.

SUMMARY OF THE INVENTION

[0014] The object of the present invention is to provide an enzymeinvolved in the xylitol biosynthesis of microorganisms excellent inxylitol production ability, genes thereof, and use thereof in order toestablish a technique for efficiently producing xylitol or breeding ofxylitol fermenting bacteria.

[0015] To achieve the aforementioned object, the present inventorssearched microorganisms having ability to directly convert D-arabitol toxylitol. As a result, they found that certain bacteria belonging to thegenus Gluconobacter or Acetobacter have such ability. Further, theysucceeded in purifying two kinds of xylitol dehydrogenase from one ofsuch bacteria, Gluconobacter oxydans, and also succeeded in isolatinggenes coding for these enzymes and determining their structures. Thus,the present invention has been accomplished.

[0016] That is, the present invention provides:

[0017] (1) a protein defined in the following (A) or (B):

[0018] (A) a protein which has the amino acid sequence of SEQ ID NO: 4in Sequence Listing;

[0019] (B) a protein which has the amino acid sequence of SEQ ID NO: 4in Sequence Listing including substitution, deletion, insertion,addition, or inversion of one or several amino acids, and has xylitoldehydrogenase activity; and

[0020] (2) a protein defined in the following (C) or (D):

[0021] (C) a protein which has the amino acid sequence of SEQ ID NO: 6in Sequence Listing;

[0022] (D) a protein which has the amino acid sequence of SEQ ID NO: 6in Sequence Listing including substitution, deletion, insertion,addition, or inversion of one or several amino acids, and has xylitoldehydrogenase activity.

[0023] The present invention also provides:

[0024] (3) a DNA which codes for a protein defined in the following (A)or (B):

[0025] (A) a protein which has the amino acid sequence of SEQ ID NO: 4in Sequence Listing;

[0026] (B) a protein which has the amino acid sequence of SEQ ID NO: 4in Sequence Listing including substitution, deletion, insertion,addition, or inversion of one or several amino acids, and has xylitoldehydrogenase activity;

[0027] (4) a DNA which codes for a protein defined in the following (C)or (D):

[0028] (C) a protein which has the amino acid sequence of SEQ ID NO: 6in Sequence Listing;

[0029] (D) a protein which has the amino acid sequence of SEQ ID NO: 6in Sequence Listing including substitution, deletion, insertion,addition, or inversion of one or several amino acids, and has xylitoldehydrogenase activity;

[0030] (5) the DNA of the above item (3), which is a DNA defined in thefollowing (a) or (b):

[0031] (a) a DNA which contains at least a nucleotide sequencecorresponding to nucleotide numbers 25 to 1053 of the nucleotidesequence of SEQ ID NO: 3 in Sequence Listing;

[0032] (b) a DNA which is hybridizable with a DNA having a nucleotidesequence corresponding to nucleotide numbers 25 to 1053 of thenucleotide sequence of SEQ ID NO: 3 in the Sequence Listing or a probeprepared from the nucleotide sequence under a stringent condition, andcodes for a protein having xylitol dehydrogenase activity;

[0033] (6) The DNA of above item (5), the stringent condition is acondition in which washing is performed at 60° C., and at a saltconcentration corresponding to 1×SSC and 0.1% SDS.

[0034] (7) the DNA of the above item (4), which is a DNA defined in thefollowing (c) or (d):

[0035] (c) a DNA which contains at least a nucleotide sequencecorresponding to nucleotide numbers 1063 to 1848 of the nucleotidesequence of SEQ ID NO: 5 in Sequence Listing;

[0036] (d) a DNA which is hybridizable with a DNA having a nucleotidesequence corresponding to nucleotide numbers 1063 to 1848 of thenucleotide sequence of SEQ ID NO: 5 in the Sequence Listing or a probeprepared from the nucleotide sequence under a stringent condition, andcodes for a protein having xylitol dehydrogenase activity; and

[0037] (8) The DNA of above item (4), the stringent condition is acondition in which washing is performed at 60° C., and at a saltconcentration corresponding to 1×SSC and 0.1% SDS.

[0038] The present invention also provides:

[0039] (9) a cell which is introduced with a DNA of any one of the aboveitems (3) to (8) in such a manner that xylitol dehydrogenase encoded bythe DNA can be expressed.

[0040] The present invention further provides:

[0041] (10) a method for producing xylitol dehydrogenase, whichcomprises cultivating the cell of the above item (9) in a medium so thatxylitol dehydrogenase should be produced and accumulated in the medium,and collecting xylitol dehydrogenase from the medium.

[0042] The present invention still further provides:

[0043] (11) a method for producing xylitol, which comprises allowingxylitol dehydrogenase of the above item (1) or (2) to act on D-xylulose,and collecting produced xylitol; and

[0044] (12) a method for producing xylitol, which comprises allowing thecell of the above item (9) to act on D-xylulose, and collecting producedxylitol.

[0045] While the xylitol dehydrogenase of the present invention hasactivity for catalyzing both of the reaction for reducing D-xylulose toproduce xylitol, and the reaction for oxidizing xylitol to produceD-xylulose, the expression “having xylitol dehydrogenase activity”herein used means to have at least the activity for catalyzing thereaction producing xylitol from D-xylulose.

[0046] According to the present invention, a novel xylitol dehydrogenaseand DNA coding for the enzyme are provided, and xylitol dehydrogenasecan be produced by using the DNA.

[0047] Further, xylitol can be produced by using a cell introduced withthe xylitol dehydrogenase or a DNA which codes for the enzyme.

[0048] Furthermore, the DNA of the present invention can be utilized forthe breeding of xylitol producing microorganisms.

BRIEF DESCRIPTION OF THE DRAWINGS

[0049]FIG. 1 is a photograph for polyacrylamide gel electrophoresis ofpurified XDH; a) CBB staining after SDS-PAGE, b) CBB staining afterNative-PAGE, and c) activity staining after Native-PAGE.

[0050]FIG. 2 is a graph representing the pH dependency of the enzymeactivity of XDH2.

DETAILED DESCRIPTION OF THE INVENTION

[0051] The present invention will be explained in detail hereafter.

[0052] <1> Xylitol dehydrogenase of the present invention

[0053] The xylitol dehydrogenase of the present invention is an enzymethat is produced by Gluconobacter oxydans. Two kinds of such enzyme werefound, one was designated as XDH1, and the other as XDH2. XDH1 has theamino acid sequence of SEQ ID NO: 4, and XDH2 has the amino acidsequence of SEQ ID NO: 6 in Sequence Listing. XDH1 and XDH2 show amolecular weight of about 36,000 to about 40,000, and about 27,000 toabout 30,000, respectively, as determined by SDS-PAGE (SDSpolyacrylamide gel electrophoresis). These two kinds of xylitoldehydrogenase, XDH1 and/or XDH2, may also be collectively referred to as“XDH” hereinafter.

[0054] As shown in Example 5 mentioned hereinafter, the optimum pH forXDH2 in the reduction reaction (reaction producing xylitol fromD-xylulose) was around 5. The optimum pH for the reduction reaction ofwell-known xylitol dehydrogenases, for example, xylitol dehydrogenasederived from Aspergillus niger is strictly 6.5 (Cor F. B. Witteveen, etal., Microbiology, 140, 1679-1685, 1994), and therefore they are clearlydifferent from XDH2 of the present invention derived from Gluconobacterbacteria in the optimum reaction pH.

[0055] As an example of the method for producing XDH of the presentinvention, methods utilized for isolation and purification of XDH fromGluconobacter oxydans will be explained below.

[0056] First, cells of Gluconobacter oxydans, for example, the strainATCC621, are disrupted by a mechanical means such as ultrasonication, oran enzymatic means utilizing a cell wall digesting enzyme etc., and acell extract is prepared by removing the insoluble fraction therefrom bycentrifugation or the like.

[0057] The cell extract obtained as described above can be fractinatedby a combination of conventional purification methods for proteins suchas anion exchange chromatography, affinity chromatography, hydrophobicchromatography, and gel filtration chromatography, to purify XDH.

[0058] As a carrier for anion exchange chromatography, Q-Sepharose FF(produced by Pharmacia), Mono-Q (produced by Pharmacia) and the like canbe mentioned. The extract containing XDH is passed through a columnfilled with such a carrier so that the enzyme should be adsorbed on thecolumn, and, after washing the column, the enzyme is eluted with abuffer of high salt concentration. In this case, the salt concentrationmay be raised stepwise, or a concentration gradient may be applied. Forexample, when Q-Sepharose FF is used, XDH adsorbed on the column may beeluted with 200 to 350 mM KCl. In the case of Mono-Q, it may be elutedwith 150 to 250 mM KCl.

[0059] As a carrier for affinity chromatography, HiTrap Blue (producedby Pharmacia) can be mentioned. The XDH of the present inventionutilizes NAD or NADH as a coenzyme, and hence has affinity for thesesubstances. XDH adsorbed on the carrier can be eluted with a buffercontaining about 5 mM NAD.

[0060] As a carrier for hydrophobic chromatography, Phenyl Sepharose HP(produced by Pharmacia) can be mentioned. XDH adsorbed on the carrier ata low salt concentration can be eluted with about 200 to 300 mM ammoniumsulfate.

[0061] The XDH purified as described above can be further purified andseparated into XDH1 and XDH2 by gel filtration chromatography, SDS-PAGEor the like. As a carrier for gel filtration chromatography, Sephadex200HP (produced by Pharmacia) can be mentioned.

[0062] In the aforementioned purification procedure, if a fractioncontains XDH or not can be confirmed by measuring the XDH activity ofthe fraction by, for example, the method shown in the examples mentionedhereinafter.

[0063] The N-terminus amino acid sequences of XDH1 and XDH2 purified asdescribed above are shown as SEQ ID NO: 1 and SEQ ID NO: 2 in SequenceListing, respectively.

[0064] While the XDH of the present invention can be obtained from cellsof Gluconobacter oxydans by isolation and purification as describedabove, it can also be produced by introducing a DNA which codes for XDHmentioned hereinafter into a suitable host so that expression of the DNAshould be obtained in accordance with a conventionally used method forproducing heterogenous proteins by fermentation.

[0065] The various genetic recombination techniques mentioned below aredescribed in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

[0066] As a host for obtaining the expression of the XDH gene, variousprokaryotic cells including Escherichia bacteria such as Escherichiacoli, Gluconobacter bacteria such as Gluconobacter oxydans, and Bacillussubtilis, and various eukaryotic cells including Saccharomycescerevisiae, Pichia stipitis and Aspergillus oryzae can be used.

[0067] A recombinant DNA used for introducing the XDH gene into a hostcan be produced by inserting a DNA coding for XDH into a vector selecteddepending on the kind of the host in which the expression is to beobtained in such a manner that the expression of XDH encoded by the DNAcan be possible. When an XDH gene specific promoter can function in thehost cell, that promoter can be used as the promoter for the expressionof the XDH gene. Further, if required, another promoter that canfunction in the host cell may be ligated to a DNA coding for XDH toobtain the expression under the control of that promoter. WhenEscherichia bacteria are used as a host, as examples of such a promoter,lac promoter, trp promoter, trc promoter, tac promoter, P_(R) promoter,P_(L) promoter of lambda phage and the like can be mentioned. As vectorsfor Escherichia bacteria, pUC19, pUC18, pBR322, pHSG299, pHSG298,pHSG399, pHSG398, RSF1010, pMW119, pMW118, pMW219, pMW218 and the likecan be mentioned. Phage DNA vectors can also be utilized. Furthermore,an expression vector containing a promoter and capable of expressing aninserted DNA sequence can also be used.

[0068]E. coli can be transformed by introducing a plasmid in accordancewith, for example, a method of D. A. Morrison (Methods in Enzymology,68, 326 (1979)) or a method in which recipient cells are treated withcalcium chloride to increase permeability for DNA (Mandel, M. and Higa,A., J. Mol. Biol., 53, 159 (1970)).

[0069] <2> DNA coding for XDH

[0070] A DNA coding for XDH can be obtained from a cDNA library orchromosome DNA library of Gluconobacter oxydans by PCR (polymerase chainreaction, see White, T. J. et al; Trends Genet., 5, 185 (1989)) orhybridization. Primers used for PCR can be designed based on the aminoacid sequences of the amino termini determined for the purified XDH1 andXDH2. Further, since the nucleotide sequences of XDH1 gene (SEQ ID NO:3) and XDH2 gene (SEQ ID NO: 5) have been elucidated according to thepresent invention, primers or probes for hybridization can be designedbased on those nucleotide sequences. By using primers having sequencecorresponding to 5′ non-translation region and 3′ non-translation regionas primers for PCR, the XDH coding region can be amplified in its fulllength. Specifically, as for XDH2, a primer having a nucleotide sequenceof a region upstream from the nucleotide number 1063 in SEQ ID NO: 5 canbe used as the 5′ primer, and a primer having a sequence complementaryto a nucleotide sequence of a region downstream from the nucleotidenumber 1851 can be used as the 3′ primer. As for XDH1, a primer having anucleotide sequence of a region upstream from the nucleotide number 25in SEQ ID NO: 3 can be used as the 5′ primer.

[0071] Synthesis of the primers can be performed by an ordinary methodsuch as a phosphoamidite method (see Tetrahedron Letters, 22, 1859(1981)) by using a commercially available DNA synthesizer (for example,DNA Synthesizer Model 380B produced by Applied Biosystems). Further, thePCR can be performed by using, for example, Gene Amp PCR System 9600produced by PERKIN ELMER and using TaKaRa LA PCR in vitro Cloning Kit(supplied by Takara Shuzo Co., Ltd.) in accordance with a methoddesignated by the suppliers.

[0072] The DNA of the present invention may code for XDH1 or XDH2including substitution, deletion, insertion, addition, or inversion ofone or several amino acids at one or a plurality of positions, providedthat the activity to produce xylitol from D-xylulose of XDH1 or XDH2encoded thereby is not deteriorated. Although the number of “several”amino acids differs depending on the position or the type of amino acidresidues in the three-dimensional structure of the protein, it may be 2to 100, preferably 2 to 50, and more preferably 2 to 10.

[0073] DNA, which codes for the substantially same protein as XDH1 orXDH2 as described above, is obtained, for example, by modifying thenucleotide sequence of XDH1 gene or XDH2 gene, for example, by means ofthe site-directed mutagenesis method so that one or more amino acidresidues at a specified site of the gene involve substitution, deletion,insertion, addition, or inversion. DNA modified as described above maybe obtained by the conventionally known mutation treatment. The mutationtreatment includes a method for treating DNA coding for XDH in vitro,for example, with hydroxylamine, and a method for treating amicroorganism, for example, a bacterium belonging to the genusEscherichia harboring DNA coding for XDH with ultraviolet irradiation ora mutating agent such as N-methyl-N′-nitro-N-nitrosoguanidine (NTG) andnitrous acid usually used for the mutation treatment.

[0074] The substitution, deletion, insertion, addition, or inversion ofnucleotide as described above also includes mutation (mutant or variant)which naturally occurs, for example, the difference in strains, speciesor genera of the microorganism.

[0075] The DNA, which codes for substantially the same protein as XDH1or XDH2, is obtained by expressing DNA having mutation as describedabove in an appropriate cell, and investigating the XDH1 or XDH2activity of an expressed product. The DNA, which codes for substantiallythe same protein as XDH1 or XDH2, is also obtained by isolating DNAwhich is hybridizable with DNA having, for example, a nucleotidesequence corresponding to nucleotide numbers of 25 to 1053 of thenucleotide sequence of SEQ ID NO: 3 or a probe which can be preparedfrom the DNA, or a nucleotide sequence corresponding to nucleotidenumbers of 1063 to 1848 of the nucleotide sequence of SEQ ID NO: 5 or aprobe which can be prepared from the DNA, under a stringent condition,and which codes for a protein having the XDH1 or XDH2 activity, from DNAcoding for XDH1 or XDH2 having mutation or from a cell harboring it. The“stringent condition” referred to herein is a condition under whichso-called specific hybrid is formed, and non-specific hybrid is notformed. It is difficult to clearly express this condition by using anynumerical value. However, for example, the stringent condition includesa condition under which DNA's having high homology, for example, DNA'shaving homology of not less than 50% are hybridized with each other, andDNA's having homology lower than the above are not hybridized with eachother. Alternatively, the stringent condition is exemplified by acondition under which DNA's are hybridized with each other at a saltconcentration corresponding to an ordinary condition of washing inSouthern hybridization, i.e., 60° C., 1×SSC, 0.1% SDS, preferably0.1×SSC, 0.1% SDS.

[0076] The gene, which is hybridizable under the condition as describedabove, includes those having a stop codon generated in the gene, andthose having no activity due to mutation of active center. However, suchmutant genes can be easily removed by ligating the gene with acommercially available activity expression vector, and measuring theXDH1 or XDH2 activity in accordance with the method described below.

[0077] A DNA which codes for the XDH of the present invention can beused for, in addition to the method for producing XDH mentioned aboveand the method for producing xylitol mentioned below, breeding ofxylitol producing microorganisms. For example, by enhancing the XDH genein a microorganism having the ability to convert D-arabitol intoxylitol, e.g., Gluconobacter bacteria, the ability of the microorganismto convert D-arabitol into xylitol can be increased.

[0078] <3> Method for producing xylitol

[0079] Xylitol can be produced by allowing the XDH of the presentinvention or a cell introduced with a DNA which codes for XDH andexpresses XDH to act on D-xylulose, and collecting produced xylitol.

[0080] XDH may be an enzyme extracted from Gluconobacter bacteria, or anenzyme produced by a genetic recombination technique utilizing a DNAwhich codes for XDH. Further, XDH may be either XDH1 or XDH2, and may bea mixture of them at an arbitrary ratio.

[0081] The reaction producing xylitol from D-xylulose usually providesgood results when performed at a temperature of 20-60° C., morepreferably 30-40° C., and pH of 4-10, more preferably pH of 4-8. For thereaction, either of standing culture or spinner culture may be used.While the reaction time may vary depending on concentration of XDH,amount of cells, and substrate concentration to be used, it is desirably1-100 hours.

[0082] For collecting and separating the produced xylitol from afinished reaction mixture, any conventional methods including use of asynthetic adsorbent, precipitant, or the like may be used.

BEST MODE FOR CARRYING OUT THE INVENTION

[0083] The present invention will be further explained more specificallywith reference to the following examples hereinafter. However, thepresent invention is not limited by the descriptions of the examples.

EXAMPLE 1 Production of XDH by Gluconobacter oxydans and PurificationThereof

[0084] <1> Culture of Gluconobacter oxydans ATCC621

[0085] The Gluconobacter oxydans strain ATCC621 was cultured to obtainits cells having sufficient XDH activity. The cultivation was alwaysperformed in PD medium as broth culture with shaking at 30° C. The PDmedium had a composition of 24 g/L potato dextrose (Difco), 30 g/L yeastextract (Difco), 5 g/L meat extract (Difco), 15 g/L glycerol, 10 g/LD-arabitol, 10 g/L D-xylose, 10 g/L xylitol, 20 g/L calcium carbonate(Kanto Chemical), pH 7.0.

[0086] First, as a seed culture, the strain ATCC621 was inoculated intoa Sakaguchi flask containing 40 ml of PD medium, and cultured overnightwith shaking at 30° C. The obtained culture broth was inoculated in anamount corresponding to 1% into 40 Sakaguchi flasks each similarlycontaining 40 ml of PD medium, and cultured with shaking for 3 days at30° C. (main culture). After removing calcium carbonate bycentrifugation, the cells were collected by centrifugation. The cellsobtained as described above were used as a material for purification ofXDH.

[0087] The XDH activity was determined by the following enzyme activityassay. 30 μl of enzyme solution was added to 570 μl of a reactionsolution containing 100 mM (final concentration) xylitol, 2 mM NAD, and100 mM CAPS (pH 10.0) to perform the enzymatic reaction at 30° C., andthe increase of NADH produced as the reaction proceeded was determinedby measuring the absorbance at 340 nm using a spectrophotometer (DU640Spectrometer produced by BECKMAN). The activity producing 1 μmol of NADHper minute was defined as 1 unit (U). The calculation was performed byusing the molecular absorption coefficient ε of NADH at 340 nm of6.3×10³.

[0088] <2> Purification of XDH

[0089] (1) Preparation of cell extract

[0090] The cells obtained above were suspended in 50 mM potassiumphosphate buffer (pH 7), and centrifuged at 5000×g for 10 minutes to becollected again in the precipitated fraction. The procedure comprisingsuspension and centrifugation of these cells was repeated twice to washthe cells.

[0091] About 10 g of the washed cells were suspended in 50 ml of Buffer1 (20 mM Tris-HCl (pH 7.6), 0.5 mM EDTA, 1 mM MgCl₂, 1 mM DTT), anddisrupted by sonication for 20 minutes at 4° C. The disrupted cellsuspension was centrifuged (8000 rpm, 10 minutes) to remove the celldebris, and further ultracentrifuged (56000 rpm, 30 minutes) to removethe insoluble fraction. Thus, a soluble fraction was obtained.

[0092] (2) Anion exchange chromatography

[0093] The above-obtained soluble fraction was loaded on an anionexchange chromatography column Q-Sepharose FF (produced by Pharmacia)equilibrated with Buffer 1. By this operation, XDH was adsorbed on thecarrier.

[0094] The protein not adsorbed on the carrier (non-adsorbed proteins)was washed off by using Buffer 1, and then the adsorbed protein waseluted by using a buffer containing KCl as an eluate. In this elution,KCl concentration in the buffer was linearly changed from 0 M to 0.5 M.The XDH activity was measured for each eluted fraction obtained by thiselution, and the XDH activity was found in eluted fractionscorresponding to the KCl concentration of about 200 to 350 mM.

[0095] (3) NAD affinity chromatography

[0096] The above-obtained fractions containing the XDH activity werecombined, and dialyzed against Buffer 1. The solution after the dialysiswas filtered through a 0.45 μm filter. The obtained filtrate was loadedon an NAD affinity column HiTrap Blue 5 ml (produced by Pharmacia)equilibrated with Buffer 1. By this operation, XDH was adsorbed on thecarrier.

[0097] The protein not adsorbed on the carrier (non-adsorbed proteins)was washed off by using Buffer 1, and then the adsorbed protein waseluted by using Buffer 2 (20 mM Tris-HCl (pH7.6), 0.5 mM EDTA, 1 mMMgCl₂, 1 mM DTT, 5 mM NAD) containing NAD as an eluate. As a result, XDHwas detected in the eluted fractions.

[0098] (4) Anion exchange chromatography

[0099] The aforementioned eluted fractions containing XDH activity werefiltered through a 0.45 μm filter. The obtained filtrate was loaded onan anion exchange chromatography column Mono-Q (produced by Pharmacia)equilibrated with Buffer 1. By this operation, XDH was adsorbed on thecarrier.

[0100] The protein not adsorbed on the carrier was washed off by usingBuffer 1, and then the adsorbed protein was eluted by using a buffercontaining KCl as an eluate. This elution was performed by linearlychanging KCl concentration in the buffer from 0 mM to 500 mM. The XDHactivity was measured for each eluted fraction obtained by this elution,and the XDH activity was found in eluted fractions corresponding to theKCl concentration of about 150 to 250 mM.

[0101] (5) Hydrophobic chromatography

[0102] The eluted fractions for which the activity was detected weredialyzed against Buffer 3 (50 mM potassium phosphate buffer, 1 Mammonium sulfate, pH 7.0). The solution obtained after the dialysis wasfiltered through a 0.45 μm filter. The obtained filtrate was loaded on ahydrophobic chromatography column Phenyl Sepharose HP (produced byPharmacia) equilibrated with Buffer 3. By this operation, XDH wasadsorbed on the carrier.

[0103] The protein not adsorbed on the carrier was washed off by usingBuffer 3, and then the adsorbed protein was eluted by using Buffer 4 (50mM potassium phosphate buffer, pH 7.0) as an eluate. For this elution,ammonium sulfate concentration in the buffer was linearly changed from 1M to 0 M. The XDH activity was measured for each eluted fractionobtained by this elution, and the XDH activity was found in elutedfractions corresponding to the ammonium sulfate concentration of about200 to 300 mM.

[0104] (6) Analysis of purified fraction

[0105] The XDH-active fraction obtained by the aforementionedpurification was subjected to SDS-PAGE and stained with CoomassieBrilliant Blue. As a result, it was confirmed that XDH had been purifiedto such a level that XDH could be detected as two bands, and theirmolecular weights were estimated to be about 27,000 to abut 30,000, andabout 37,000 to about 40,000, respectively (see FIG. 1). Henceforth, theprotein corresponding to the band of molecular weight of about 36,000 toabut 40,000 is referred to as XDH1, and the protein corresponding to theband of molecular weight of about 27,000 to about 30,000 as XDH2.

[0106] Further, the obtained active fraction was subjected toNative-PAGE (non-denaturation PAGE), and stained with CoomassieBrilliant Blue. As a result, two bands corresponding to molecularweights of more than 100 kDa were confirmed. When the gel afterNative-PAGE was subjected to activity staining with an activity stainingsolution (25 mM glycine buffer, 2.5 mM NAD, 50 mM xylitol, 0.2 nMphenazine methosulfate, 0.2 mM tetranitro blue tetrazolium chloride),the XDH activity was detected in both of the two corresponding bands,and it was confirmed that both of the proteins corresponding to the twobands detected in the SDS-PAGE had XDH activity (FIG. 1). The purifiedfraction containing these XDH1 and XDH2 will sometimes be referred tosimply as XDH hereinafter.

[0107] The increase of the XDH specific activity as a result of theaforementioned purification was determined. The XDH activity of theaforementioned cell extract and the active fraction obtained by thepurification was measured. As a result, it was found that the specificactivity per unit protein weight was increased by about 550 times by theseries of purification procedures. By the activity assay method used forthis measurement, the specific activity of the purified XDH wasestimated to be about 130 U/mg (30° C., pH 10).

[0108] (7) Determination of amino acid sequence at amino terminus of XDH

[0109] The N-terminus sequence of the XDH purified as described abovewas sequenced as follows. That is, about 10 μg in terms of protein ofthe purified XDH fraction was electrophoresed in polyacrylamide gel inthe presence of SDS, and then the XDH in the gel was blotted to amembrane filter, and analyzed for the amino acid sequence from theN-terminus by a protein sequencer. Specifically, the objective enzymeswere blotted to a polyvinylidene fluoride (PVDF) membrane from the gelafter the electrophoresis by the semi-dry method (Tanpakushitu KozoKaiseki [Analysis of Protein Structure], H. Hirano, Tokyo Kagaku Dojin)by using Milliblot (Millipore). Then, the objective enzymes (XDH1 andXDH2) on the PVDF film were analyzed by a protein sequencer (Model 476Aproduced by ABI) to perform N-terminus amino acid sequence analysis.

[0110] As a result, the amino acid sequence of 27 residues from theN-terminus was determined for XDH1, and the amino acid sequence of 25residues from the N-terminus was determined for XDH2. The amino acidsequence of the determined N-terminus sequence of XDH1 was shown as SEQID NO: 1 in Sequence Listing, and the amino acid sequence of theN-terminus sequence of XDH2 was shown as SEQ ID NO: 2 in SequenceListing, respectively.

EXAMPLE 2 Conversion of D-xylulose into Xylitol by XDH

[0111] D-Xylulose was converted into xylitol using the purified XDH(XDH1 and XDH2) obtained in Example 1. 0.2 U of the purified XDH wasadded to 0.25 ml of a reaction solution containing 21 mM D-xylulose, 20mM NADH, and 100 mM Tris-HCl buffer (pH 8.0), and incubated at 30° C.for 1 hour to allow the reaction. The solution after the reaction wassubjected to high performance liquid chromatography (HPLC) to analyzethe produced xylitol under the following conditions.

[0112] Column: Shodex SC1211 (produced by Showa Denko Co., Ltd.)

[0113] Mobile phase: 50% acetonitrile/50% 50 ppm aqueous Ca-EDTA

[0114] Flow rate: 0.8 ml/minute

[0115] Temperature: 60° C.

[0116] Detection: RI detector

[0117] As a result, formation of 18 mM xylitol was observed in thesolution after the reaction, and it was shown that xylitol could beproduced from D-xylulose using the purified XDH.

EXAMPLE 3 Isolation of XDH Gene Derived from Gluconobacter

[0118] <1> Amplification of XDH gene fragment by PCR

[0119] (1) Preparation of PCR primers based on N-terminus amino acidsequence of XDH

[0120] Based on each of the aforementioned N-terminus amino acidsequences (SEQ ID NOS: 1 and 2) of XDH (XDH1, XDH2) derived fromGluconobacter oxydans ATCC621, mixed primers which had the nucleotidesequences shown as SEQ ID NO: 7-10, respectively, were prepared.

[0121] (2) Preparation of chromosome DNA of Gluconobacter oxydansATCC621

[0122]Gluconobacter oxydans ATCC621 strain was cultured under thefollowing conditions. First, the ATCC621 strain was cultured in 20 ml ofYPG medium (3% glucose, 0.5% Bacto yeast extract, 0.3% Bacto peptone, pH6.5) overnight as a seed culture. By using 5 ml of this culture as seedbacteria, main culture was performed using 100 ml of YPG medium. Theculture was performed with shaking at 30° C.

[0123] After the bacteria were cultured to late log phase under theaforementioned conditions, 100 ml of the culture broth was centrifuged(12000×g, 4° C., 15 minutes) to collect the cells. The cells weresuspended in 10 ml of 50:20 TE (50 mM Tris-HCl, pH 8.0, 20 mM EDTA), andthe cells were washed and recovered by centrifugation. The cells weresuspended in 10 ml of 50:20 TE again. To this suspension, 0.5 ml of 20mg/ml lysozyme solution and 1 ml of 10% SDS solution were added, andincubated at 55° C. for 20 minutes. After the incubation,deproteinization was performed by adding equal volume of 10:1TE-saturated phenol. DNA was precipitated by adding equal volume of2-propanol to the separated aqueous layer, and collected. Theprecipitated DNA was dissolved in 0.5 ml of 50:20 TE, added with 5 μl of10 mg/ml RNase and 5 μl of 10 mg/ml Proteinase K, and allowed to reactat 55° C. for 2 hours. After the reaction, deproteinization wasperformed by adding equal volume of 10:1 TE-saturated phenol. Theseparated aqueous layer was further added with equal volume of 24:1chloroform/isoamyl alcohol, and stirred, and the aqueous layer wascollected. After this procedure was further repeated twice, the obtainedaqueous layer was added with 3 M sodium acetate solution (pH 5.2) sothat a final concentration of 0.4 M should be obtained, and furtheradded with twice as much volume of ethanol. The produced DNA wascollected as precipitates, washed with 70% ethanol, dried, and dissolvedin 1 ml of 10:1 TE.

[0124] (3) Preparation of DNA fragment by PCR

[0125] The DNA molecule containing the gene coding for XDH derived fromGluconobacter bacteria was amplified and isolated by PCR using TaKaRa LAPCR in vitro Cloning Kit (supplied by Takara Shuzo Co., Ltd.). Theexperiments were performed according to the instruction attached to thekit hereafter unless otherwise indicated.

[0126] Five μg of the chromosome DNA produced as described in the above(2) was digested with restriction enzymes PstI or HindIII respectively.Then, a PstI cassette or HindIII cassette was ligated to the DNAfragments collected by the ethanol precipitation. Furthermore, afterperforming ethanol precipitation, first PCR was performed for thecollected DNA by using a combination of primers of the primer C1 andprimers mentioned below. That is, a DNA ligated to PstI cassette wasused as a template DNA for the primer XDH1-S1 that was based on theamino acid sequence of XDH1, and a DNA ligated to HindIII cassette wasused as a template DNA for the primer XDH2-S1 that was based on thesequence of XDH2, respectively. There are shown the nucleotide sequencesof the primer C1, the primer XDH1-S1, and the primer XDH2-S1 as SEQ IDNO: 11, SEQ ID NO: 7, and SEQ ID NO: 9 in Sequence Listing,respectively. The primer C1 was contained in the TaKaRa LA PCR in vitroCloning Kit, and corresponded to the sequence in the PstI cassette andthe HindIII cassette. The PCR reaction was performed by using Gene AmpPCR System 9600 (produced by PERKIN ELMER), and a reaction according tothe following conditions was repeated for 30 cycles.

[0127] 94° C. for 30 seconds,

[0128] 55° C. for 2 minutes

[0129] 72° C. for 1 minute

[0130] Then, the reaction mixture was diluted 100 times, and newly addedwith the primer C2 and the primer XDH1-S2 or the primer XDH2-S2, and thesecond PCR was performed. The conditions were the same as those of thefirst PCR. The nucleotide sequences of the primer C2, the primerXDH1-S2, and the primer XDH2-S2 are shown as SEQ ID NO: 12, SEQ ID NO:8, and SEQ ID NO: 10 in Sequence Listing, respectively. The primer C2was contained in the TaKaRa LA PCR in vitro Cloning Kit, and had thesequence corresponding to the sequence in the PstI cassette and theHindIII cassette. The primer XDH1-S2 and the primer XDH2-S2 eachcontained a sequence designed based on the amino acid sequencesdetermined, the sequence corresponding to EcoRI site, and EcoRI site.

[0131] After the reaction, 3 μl of the reaction mixture was subjected to0.8% agarose gel electrophoresis. As a result, it was confirmed that aDNA fragment of about 1 kb was amplified when primer XDH1-S2 was used,and a DNA fragment of about 1.7 kb was amplified when XDH2-S2 was used.

[0132] (4) Cloning of DNA fragments amplified by PCR into pUC19

[0133] Cloning was performed by ligating the DNA fragments of about 1kbp (XDH1) and about 1.7 kbp (XDH2) amplified by PCR with pUC19. Theligation was performed by using DNA Ligation Kit Ver.2 (supplied byTakara Shuzo Co., Ltd.). The experiments were performed according to theinstruction attached to the kit hereafter unless otherwise indicated.

[0134] 400 ng of the DNA fragment of about 1 kb, which had beenamplified by using the primer XDH1-S2, was digested with PstI and EcoRI,then purified, and ligated to pUC19 digested with PstI and EcoRI.Escherichia coli JM109 was transformed by using this ligation reactionmixture.

[0135] Further, 400 ng of the DNA fragment of about 1.7 kb, which hadbeen amplified by using the primer XDH2-S2, was digested with HindIIIand EcoRI, then purified, and ligated to pUC19 digested with HindIII andEcoRI. Escherichia coli JM109 was transformed by using this ligationreaction mixture.

[0136] From the obtained transformant cells, several JM109 strainstransformed with pUC19 and containing the target DNA fragment of about 1kbp (XDH1) or about 1.7 kbp (XDH2) were selected for each case. Theselection was performed according to the method described in MolecularCloning, 2nd edition, Cold Spring Harbor Press (1989).

[0137] (5) Determination of nucleotide sequence of XDH2 gene fragment

[0138] The plasmid carried by JM109 transformed with pUC19 containingthe DNA fragment of about 1.7 kbp (XDH2) was prepared according to themethod described in Molecular Cloning, 2nd edition, Cold Spring HarborPress (1989), and the nucleotide sequence of the inserted DNA fragmentwas determined. The sequencing reaction was performed by using DyeTerminator Cycle Sequencing Kit (produced by ABI) according to theinstruction attached to the kit. The electrophoresis was performed byusing DNA Sequencer 373 (produced by ABI).

[0139] As a result, it was found that the DNA fragment amplified by PCRhad a sequence of from the thymidine residue at position 1116 to thethymidine residue at position 2774 of the nucleotide sequence shown asSEQ ID NO: 5 in Sequence Listing.

[0140] (6) Preparation of DNA fragment of upstream region of XDH2 geneby PCR

[0141] The XDH2 gene and a DNA fragment of the upstream region of theXDH2 gene were amplified and isolated by PCR using the nucleotidesequences determined above. The PCR reaction was performed by usingTaKaRa LA PCR in vitro Cloning Kit (supplied by Takara Shuzo Co., Ltd.).The experiments were performed according to the instruction attached tothe kit hereafter unless otherwise indicated.

[0142] Five μg of the chromosome DNA prepared as in the above (2) wasdigested with restriction enzyme SalI. Then, SalI cassette was ligatedto the DNA fragment collected by ethanol precipitation. Ethanolprecipitation was further performed, and first PCR was performed for thecollected DNA by using the primer C1 and the primer XDH2UP-S1. Thenucleotide sequences of the primer C1 and the primer XDH2UP-S1 are shownas SEQ ID NO: 11 and SEQ ID NO: 13 in Sequence Listing, respectively.The primer XDH2UP-S1 is a sequence complementary to the region of fromthe cytosine residue at position 1317 to the cytosine residue atposition 1283 of the nucleotide sequence of the gene cluster coding forXDH2 of Gluconobacter shown as SEQ ID NO: 5.

[0143] The PCR reaction was performed by using Gene Amp PCR System 9600(produced by PERKIN ELMER), and a reaction according to the followingconditions was repeated for 30 cycles.

[0144] 94° C. for 30 seconds,

[0145] 55° C. for 2 minutes

[0146] 72° C. for 1 minute

[0147] Then, the reaction mixture was diluted 100 times, and newly addedwith the primer C2 and the primer XDH2UP-S2 to perform the second PCR.The conditions were the same as those of the first PCR. The sequences ofthe primer C2 and the primer XDH2UP-S2 are shown as SEQ ID NO: 12 andSEQ ID NO: 14 in Sequence Listing, respectively. The primer XDH2UP-S2 iscomposed of a sequence complementary to the region of from the guanosineresidue at position 1255 to the guanosine residue at position 1225 ofthe nucleotide sequence of the gene coding for XDH2 of Gluconobactershown as SEQ ID NO: 5. After the reaction, 3 μl of the reaction mixtureswas subjected to 0.8% agarose gel electrophoresis. As a result, it wasconfirmed that a DNA fragment of about 1.3 kb had been amplified.

[0148] (7) Determination of nucleotide sequence of XDH2 gene and DNAfragment containing upstream region thereof

[0149] The DNA fragment of about 1.3 kbp amplified by the aforementionedPCR was purified, and determined for the nucleotide sequence. Thesequencing reaction was performed by using Dye Terminator CycleSequencing Kit (produced by ABI) according to the instruction attachedto the kit. The electrophoresis was performed by using DNA Sequencer 373(produced by ABI).

[0150] As a result, it was found that the DNA fragment amplified in theabove (6) had a sequence from the guanosine residue at position 1 to theguanosine residue at position 1224 of the nucleotide sequence shown asSEQ ID NO: 5 in Sequence Listing. The nucleotide sequence shown in SEQID NO: 5 comprises this nucleotide sequence combined with the nucleotidesequence determined in the above (5). The amino acid sequence which maybe encoded by this nucleotide sequence, deduced based on the universalcodons, is shown together in SEQ ID NO: 5, and also shown as SEQ ID NO:6. The sequence of from 2nd to 26th amino acid residues of that aminoacid sequence completely corresponded to the sequence of the 1st to the25th amino acid residues of the N-terminus amino acid sequence of XDH2shown as SEQ ID NO: 2. From this, it was confirmed that the DNAfragments amplified by the PCR were the target XDH2 gene and itsupstream region derived from Gluconobacter bacteria.

[0151] (8) Cloning of DNA fragment containing full length XDH2 genecoding region

[0152] Cloning was performed by amplifying a DNA fragment containingfull length XDH2 gene coding region by PCR, and ligating it to pUC18.The PCR reaction was performed by using TaKaRa LAPCR kit (supplied byTakara Shuzo Co., Ltd.). The experiments were performed according to theinstruction attached to the kit hereafter unless otherwise indicated.

[0153] PCR was performed by using 1 μg of chromosome DNA ofGluconobacter oxydans ATCC621 strain produced in the same manner as inthe above (2) as template, and using a primer XDH2-5′ and a primerXDH2-3′. The nucleotide sequence of the primer XDH2-5′ was shown as SEQID NO: 15, and the nucleotide sequence of the primer XDH2-3′ was shownas SEQ ID NO: 16 in Sequence Listing. The primer XDH2-5′ comprises asequence corresponding to the region from the cytosine residue atposition 1043 to the adenosine residue at position 1063 of thenucleotide sequence containing the XDH2 gene shown as SEQ ID NO: 5, andthe primer XDH2-3′ comprises a sequence complementary to the region fromthe guanosine residue at position 1957 to the cytosine residue atposition 1978 of the same.

[0154] The PCR reaction was performed by using GeneAmp PCR System 9600(produced by PERKIN ELMER), and a reaction according to the followingconditions was repeated for 30 cycles.

[0155] 94° C. for 30 seconds,

[0156] 55° C. for 2 minutes

[0157] 72° C. for 1 minute

[0158] After the reaction, 3 μl of the reaction mixture was subjected to0.8% agarose gel electrophoresis. As a result, it was confirmed that aDNA fragment of about 1 kbp had been amplified.

[0159] The DNA fragment of about 1 kbp amplified by the aforementionedPCR was ligated to pUC18 to perform cloning. The cloning was performedby using DNA Ligation Kit Ver.2 (supplied by Takara Shuzo Co., Ltd.).The experiments were performed according to the instruction attached tothe kit hereafter unless otherwise indicated. 400 ng of the amplifiedDNA fragment of about 1 kb was digested with BamHI and EcoRI, thenpurified, and ligated to pUC18 digested with BamHI and EcoRI.Escherichia coli JM109 was transformed by using this ligation reactionmixture.

[0160] From the obtained transformants, several JM109 strainstransformed with pUC18 containing the target DNA fragment of about 1 kbpwere selected. The selection was performed according to the methoddescribed in Molecular Cloning, 2nd edition, Cold Spring Harbor Press(1989).

[0161] The XDH2 gene derived form Gluconobacter oxydans could be clonedas described above. The plasmid having the target XDH2 gene fragmentobtained by the aforementioned method is referred to as pUCXDH2.

[0162] (9) Determination of nucleotide sequence of XDH1 gene fragment

[0163] The plasmid carried by the JM109 strains transformed with pUC18containing the DNA fragment of about 1 kbp (XDH1) and selected above (4)was extracted according to the method described in Molecular Cloning,2nd edition, Cold Spring Harbor Press (1989), and the nucleotidesequence of the inserted DNA fragment was determined. The sequencingreaction was performed by using Dye Terminator Cycle Sequencing Kit(produced by ABI) according to the instruction attached to the kit. Theelectrophoresis was performed by using DNA Sequencer 373 (produced byABI).

[0164] As a result, it was found that the DNA fragment amplified by PCRhad a sequence from the guanosine residue at position 52 to theguanosine residue at position 1011 of the nucleotide sequence shown asSEQ ID NO: 3 in Sequence Listing.

[0165] (10) Cloning of XDH1 gene from chromosome DNA library

[0166] i) Construction of chromosome DNA library

[0167] One μg of the chromosome DNA prepared in the above (2) wascompletely digested with HindIII. After the DNA was collected by ethanolprecipitation, it was dissolved in 10 μl of 10:1 TE. Five μg of thissolution and 1 ng of pUC19 (supplied by Takara Shuzo Co., Ltd.) whichhad been digested with HindIII and subjected to dephosphorylation withBAP (bacterial alkaline phosphatase) were mixed, and the ligationreaction was performed by using DNA Ligation Kit Ver.2 (supplied byTakara Shuzo Co., Ltd.). Three μl of this ligation reaction mixture wasmixed with 100 μl of competent cells of Escherichia coli JM109 strain(supplied by Takara Shuzo Co., Ltd.) to transform the Escherichia coliJM109 strain. This was applied on a suitable solid medium to create achromosome DNA library.

[0168] ii) Preparation of probe

[0169] It was decided to use a part of the XDH1 gene obtained in theabove (3) for a probe. The DNA fragment of about 1 kb amplified by usingthe primer C2 and the primer XDH1-S2, which was obtained in the above(3), was separated by 1% agarose gel electrophoresis. The target bandwas excised, and the DNA was purified by using Gene Clean II Kit(produced by Funakoshi). Finally, 16 μl of 50 ng/μl DNA solution wasobtained. A probe labeled with digoxigenin was obtained by using thisDNA fragment and DIG High Prime (produced by Boehringer Mannheim)according to the instruction attached to the product.

[0170] iii) Screening by colony hybridization

[0171] In order to obtain the XDH1 gene in full length, screening of thechromosome DNA library by colony hybridization utilizing theaforementioned probe was performed. The colony hybridization wasperformed according to the method described in Molecular Cloning, 2ndedition, Cold Spring Harbor Press (1989).

[0172] The colonies of the chromosome DNA library were blotted to anylon membrane filter (Hybond-N produced by Amersham), denatured withalkali, neutralized, and immobilized. Hybridization was performed byusing EASY HYB (produced by Boehringer Mannheim). The filter wasimmersed into the buffer (EASY HYB), and pre-hybridization was performedat 42° C. for one hour. Then, the labeled probe produced above wasadded, and hybridization was performed at 42° C. for 16 hours. Then, thefilter was washed with 2×SSC containing 0.1% SDS at room temperature for20 minutes. Further, it was washed twice with 0.1×SSC containing 0.1%SDS at 65° C. for 15 minutes.

[0173] The colonies hybridizable with the probe was detected by usingDIG Nucleotide Detection Kit (produced by Boehringer Mannheim) accordingto the instruction attached to the kit. As a result, four strains ofcolonies hybridizable with the probe could be confirmed.

[0174] (11) DNA sequence of XDH1 gene

[0175] In the same manner as in the above (5), the nucleotide sequenceof the DNA fragment inserted into pUC19 was determined. The result isshown as SEQ ID NO: 3 in Sequence Listing. The amino acid sequence,which is deduced to be encoded by the nucleotide sequence based onuniversal codons, is shown in SEQ ID NO: 3 together with the nucleotidesequence, and also shown as SEQ ID NO: 4. The amino acid sequence fromthe 2nd to 28th amino acid residues completely corresponded to thesequence composed of the 27 residues of the 1st to the 27th amino acidresidues of the sequence shown as SEQ ID NO: 1. From this, it wasconfirmed that the obtained DNA fragment was the target XDH1 genederived from Gluconobacter bacteria and flanking regions thereof.

EXAMPLE 4 Expression of XDH2 Gene Derived from Gluconobacter Bacteria inEscherichia coli and Purification of the Product

[0176] <1> Culture of Escherichia coli harboring recombinant XDH2 geneand induction of expression

[0177] In the pUCXDH2 obtained in Example 3, the DNA coding for XDH2gene derived from Gluconobacter bacteria is ligated downstream of lacZpromoter, and therefore it was designed to be expressed under thecontrol of lacZ promoter.

[0178]Escherichia coli JM109 transformed with pUCXDH2, and Escherichiacoli JM109 transformed with pUC18 as a control were cultured at 37° C.overnight with shaking in 50 ml of LB medium containing 100 μg/ml ofampicillin. These were used as seed culture. The seed culture ofEscherichia coli JM109 transformed with pUCXDH2 was inoculated in anamount of 1% to a flask containing fresh medium, and this was designatedas Experimental panel 1. On the other hand, the seed culture ofEscherichia coli JM109 transformed with pUC18 was similarly inoculatedin an amount of 1% to a flask, and this was designated as Experimentalpanel 2 (control). Each experimental panel was cultured, and whenabsorbance of the culture for a light having a wavelength of 610 nmbecame about 0.7, it was added with IPTG(isopropyl-beta-D-thiogalactopyranoside) to a final concentration of 1mM. Then, after 4 hours, the culture was completed.

[0179] <2> Confirmation of protein obtained by induced expression

[0180] After the completion of the cultivation, the cells were collectedby centrifugation (12,000×g, 15 minutes) of 10 ml of the culture broth.The cells were suspended in 2 ml of 10 mM Tris-HCl, pH 7.5, washed andrecovered by centrifugation. The cells were suspended in 1 ml of thesame buffer, and disrupted by shaking with 0.1 mm zirconia beads for 3minutes using Multi Beads Shocker (Yasui Kikai). This disrupted cellsuspension was subjected to SDS-PAGE, and stained with CBB (CoomassieBrilliant Blue). As a result, a band corresponding to a molecular weightof about 27,000 to 30,000 was confirmed, which was observed only inExperimental panel 1 (JM109 transformed with pUCXDH2). Deduced from themolecular weight, it was considered that the desired XDH2 protein wasexpressed.

[0181] <3> Confirmation of XDH activity

[0182] The XDH activity of the expressed protein was measured. The XDHactivity was measured by using the aforementioned disrupted cellsuspension according to the method described in Example 1. As a result,14 U/mg of the XDH activity was detected in the Escherichia coli JM109transformed with pUCXDH2, whereas no XDH activity was detected in theEscherichia coli JM109 transformed with pUC18 as the control. From thisresult, it was confirmed that Escherichia coli JM109 transformed withpUCXDH2 showed the XDH activity.

[0183] <4> Purification of XDH2 from recombinant Escherichia coli JM109

[0184] The Escherichia coli JM109 cells transformed with pUCXDH2, whichwere cultured in the above <2>, were collected by centrifugation. Theobtained cells were used as a material for purification of XDH. The XDHactivity was measured by the method described in Example 1.

[0185] (1) Preparation of cell extract

[0186] The above bacterial cells were suspended in 50 mM potassiumphosphate buffer (pH 7), and collected again in a precipitated fractionobtained by centrifugation at 5000×g for 10 minutes. This procedurecomprising suspension and centrifugation was performed as washing of thecells. This washing process of the cells was repeated twice.

[0187] Three grams of the washed cells was suspended in 20 ml of Buffer1 (20 mM Tris-HCl (pH 7.6), 0.5 mM EDTA, 1 mM MgCl₂, 1 mM DTT), anddisrupted by sonication for 20 minutes at 4° C. The disrupted suspensionwas centrifuged (8000 rpm, 10 minutes) to remove cell residues, andultracentrifuged (56000 rpm, 30 minutes) to remove insoluble fraction.

[0188] (2) Anion exchange chromatography

[0189] The obtained soluble fraction was loaded on an anion exchangechromatography column Q-Sepharose FF (produced by Pharmacia)equilibrated by Buffer 1. By this operation, XDH was adsorbed on thecarrier.

[0190] The protein not adsorbed on the carrier (non-adsorbed protein)was washed off by using Buffer 1, and then the adsorbed protein waseluted by using a buffer containing KCl as an eluate. In this elution,KCl concentration in the buffer was linearly changed from 0 M to 0.5 M.The XDH activity was measured for each eluted fraction obtained by thiselution, and the XDH activity was found in eluted fractionscorresponding to the KCl concentration of about 200 to 350 mM.

[0191] (3) NAD affinity chromatography

[0192] The above-obtained fractions containing the XDH activity werecombined, and dialyzed against Buffer 1. The solution after the dialysiswas filtered through a 0.45 μm filter. The obtained filtrate was loadedon an NAD affinity column HiTrap Blue 5 ml (produced by Pharmacia)equilibrated with Buffer 1. By this operation, XDH was adsorbed on thecarrier. Then, the protein not adsorbed on the carrier (non-adsorbedprotein) was washed off by using Buffer 1, and then the adsorbed proteinwas eluted by using, as an eluate, Buffer 2 (20 mM Tris-HCl (pH 7.6),0.5 mM EDTA, 1 mM MgCl₂, 1 mM DTT, 5 mM NAD) containing NAD. As aresult, XDH was detected in the eluted fractions.

[0193] (4) Hydrophobic chromatography

[0194] The eluted fractions for which the activity was detected weredialyzed against Buffer 3 (50 mM potassium phosphate buffer, 1 Mammonium sulfate, pH 7.0). The solution obtained after the dialysis wasfiltered through a 0.45 μm filter. The obtained filtrate was loaded on ahydrophobic chromatography column Phenyl Sepharose HP (produced byPharmacia) equilibrated with Buffer 3. By this operation, XDH wasadsorbed on the carrier.

[0195] Then, the protein not adsorbed on the carrier was washed off byusing Buffer 3, and then the adsorbed protein was eluted by using Buffer4 (50 mM potassium phosphate buffer, pH 7.0) as an eluate. For thiselution, ammonium sulfate concentration in the buffer was linearlychanged from 1 M to 0 M. The XDH activity was measured for each elutedfraction obtained by this elution, and the XDH activity was found ineluted fractions corresponding to the ammonium sulfate concentration ofabout 200 to 300 mM.

[0196] The obtained active fraction was subjected to SDS-PAGE, andstained with Coomassie Brilliant Blue. As a result, it was confirmedthat XDH2 had been purified to such a level that XDH2 could be detectedas a single band, and its molecular weight was estimated to be about27,000 to abut 30,000. That is, XDH2 expressed in Escherichia coli JM109could be purified as a single enzyme.

EXAMPLE 5 Determination of Optimum pH of XDH

[0197] By using the XDH2 enzyme obtained in Example 4, variation of theenzyme activity depending on the reaction pH was measured as follows todetermine the optimum pH.

[0198] Sodium acetate buffers (pH 3.3, 4, 4.5, 5 and 6), Tris-HCl (pH 7and 8), Glycine-NaOH (pH 9), and CAPS-NaOH (pH 10) buffers were used forthe enzyme reaction buffers. Measurement of the XDH activity for thereduction reaction was performed as follows. Thirty μl of an enzymesolution was added to 570 μl of a reaction solution containing 100 mM(final concentration) D-xylulose, 0.2 mM NADH, and 100 mM buffer toallow the enzymatic reaction at 30° C., and the decrease of NADH causedby the reaction was determined by measuring the absorbance at 340 nmusing a spectrophotometer (DU 640 Spectrometer produced by BECKMAN). Theactivity decreasing 1 μmol of NADH per minute was defined as 1 U. Thecalculation was performed by using the molecular extinction coefficientε of NADH at 340 nm of 6.3×10³. Each buffer was added so that it shouldhave a concentration of 100 mM in the reaction solution. The XDHfraction purified above was used as an enzyme source, and the reactionwas performed at 30° C. The result of the measurement was represented asa relative value of enzyme activity to the actually determined pH valueof each reaction solution. For convenience, the activity for theoxidation reaction at pH 5 was defined as 100. The results of themeasurement are shown in FIG. 2.

[0199] It was found that the optimum pH for the reduction reaction(reaction producing xylitol from D-xylulose) of the XDH2 of the presentinvention was about 5 (see FIG. 2). Since the optimum pH for thereduction reaction of the XDH derived from Aspergillus niger reported byCor F. B. Witteveen, et al. (Microbiology, 140, 1679-1685, 1994) isstrictly 6.5, and therefore the XDH2 of the present invention derivedfrom Gluconobacter bacteria is clearly different from the known XDH inthe reaction optimum pH. That is, it was demonstrated that, among theGluconobacter bacteria derived XDH found by the present invention, atleast XDH2 was characterized in that it had a lower optimum pH for thereduction reaction.

What is claimed is:
 1. A protein defined in the following (A) or (B):(A) a protein which has the amino acid sequence of SEQ ID NO: 4; (B) aprotein which has the amino acid sequence of SEQ ID NO: 4 in SequenceListing including substitution, deletion, insertion, addition, orinversion of one or several amino acids, and has xylitol dehydrogenaseactivity.
 2. A protein defined in the following (C) or (D): (C) aprotein which has the amino acid sequence of SEQ ID NO: 6; (D) a proteinwhich has the amino acid sequence of SEQ ID NO: 6 includingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids, and has xylitol dehydrogenase activity.
 3. A DNAwhich codes for a protein defined in the following (A) or (B): (A) aprotein which has the amino acid sequence of SEQ ID NO: 4; (B) a proteinwhich has the amino acid sequence of SEQ ID NO: 4 includingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids, and has xylitol dehydrogenase activity.
 4. A DNAwhich codes for a protein defined in the following (C) or (D): (C) aprotein which has the amino acid sequence of SEQ ID NO: 6; (D) a proteinwhich has the amino acid sequence of SEQ ID NO: 6 includingsubstitution, deletion, insertion, addition, or inversion of one orseveral amino acids, and has xylitol dehydrogenase activity.
 5. The DNAof claim 3 , which is a DNA defined in the following (a) or (b): (a) aDNA which contains at least a nucleotide sequence corresponding tonucleotide numbers 25 to 1053 of the nucleotide sequence of SEQ ID NO:3; (b) a DNA which is hybridizable with a DNA having a nucleotidesequence corresponding to nucleotide numbers 25 to 1053 of thenucleotide sequence of SEQ ID NO: 3 or a probe prepared from thenucleotide sequence under a stringent condition, and codes for a proteinhaving xylitol dehydrogenase activity.
 6. The DNA of claim 5 , thestringent condition is a condition in which washing is performed at 60°C., and at a salt concentration corresponding to 1×SSC and 0.1% SDS. 7.The DNA of the claim 4 , which is a DNA defined in the following (c) or(d): (c) a DNA which contains at least a nucleotide sequencecorresponding to nucleotide numbers 1063 to 1848 of the nucleotidesequence of SEQ ID NO: 5; (d) a DNA which is hybridizable with a DNAhaving a nucleotide sequence corresponding to nucleotide numbers 1063 to1848 of the nucleotide sequence of SEQ ID NO: 5 or a probe prepared fromthe nucleotide sequence under a stringent condition, and codes for aprotein having xylitol dehydrogenase activity.
 8. The DNA of claim 7 ,the stringent condition is a condition in which washing is performed at60° C., and at a salt concentration corresponding to 1×SSC and 0.1% SDS.9. A cell which is introduced with a DNA of any one of claims 3 to 8 insuch a manner that xylitol dehydrogenase encoded by the DNA can beexpressed.
 10. A method for producing xylitol dehydrogenase, whichcomprises cultivating the cell of claim 9 in a medium so that xylitoldehydrogenase should be produced and accumulated in the medium, andcollecting xylitol dehydrogenase from the medium.
 11. A method forproducing xylitol, which comprises allowing xylitol dehydrogenase ofclaim 1 or 2 to act on D-xylulose, and collecting produced xylitol. 12.A method for producing xylitol, which comprises allowing the cell ofclaim 9 to act on D-xylulose, and collecting produced xylitol.